Journal: Journal of Virology

Article Title: Evasion of the Innate Immune Type I Interferon System by Monkeypox Virus

doi: 10.1128/JVI.00304-15

Figure Lengend Snippet: MPXV replication in the presence of IFN. (A) RK13 cells were treated with increasing amounts of IFN-α A/D for 18 h and then infected with 100 PFU of MPXV. Cells were stained at 48 hpi with crystal violet. (B) Subconfluent BSC-40 cells were treated with the indicated amount of recombinant IFN for 18 h prior to infection. Treated cells were infected with approximately 100 PFU of VACV, EMCV, or MPXV. Cells were stained with crystal violet at 48 hpi.

Article Snippet: BSC-40 and RK13 cells were seeded in 6-well plates and pretreated with 0, 1, 10, 100, or 1,000 IU/ml of IFN-α A/D (cross-reactive with human, monkey, mouse, bovine, rat, cat, pig, rabbit, guinea pig, or hamster cells; catalog number 11200-2; PBL).

Techniques: Infection, Staining, Recombinant

Journal: Journal of Virology

Article Title: Evasion of the Innate Immune Type I Interferon System by Monkeypox Virus

doi: 10.1128/JVI.00304-15

Figure Lengend Snippet: The MPXV F3 protein restores the IFNr phenotype. (A) Through in vitro recombination, the F3L gene of MPXV was inserted into the E3L locus of VACVΔE3L, generating a recombinant VACV that expresses the MPXV F3 protein (VACV-F3L). RK13 cells were treated with increasing amounts of IFN-α A/D for 18 h, prior to infection with 100 PFU of VACV-F3L. At 48 hpi, the cells were stained with crystal violet. (B) Subconfluent BSC-40 cells were treated with the indicated amount of recombinant IFN at 18 hpi. Treated cells were infected with approximately 100 PFU of VACV E3LΔ37N, VACV-F3L, or EMCV. Cells were stained with crystal violet at 48 hpi.

Article Snippet: BSC-40 and RK13 cells were seeded in 6-well plates and pretreated with 0, 1, 10, 100, or 1,000 IU/ml of IFN-α A/D (cross-reactive with human, monkey, mouse, bovine, rat, cat, pig, rabbit, guinea pig, or hamster cells; catalog number 11200-2; PBL).

Techniques: In Vitro, Recombinant, Infection, Staining

Journal: bioRxiv

Article Title: Distinct SARS-CoV-2 sensing pathways in pDCs driving TLR7-antiviral vs. TLR2-immunopathological responses in COVID-19

doi: 10.1101/2021.11.23.469755

Figure Lengend Snippet: pDCs were either mock treated or exposed to the SARS-CoV-2 FR2020 early Wuhan-like strain or the SARS-CoV-2 alpha variant B.1.1.7 (0.1 MOI). Supernatants were collected at indicated time points and the production of type I IFNα (A) and CXCL10 (B) was quantified. The FR2020 strain was used in subsequent experiments where pDC were either mock treated (mock, grey), exposed to SARS-CoV-2 at 1 MOI (SARS-2, purple), TLR7 (2.5 μg/mL R837, blue) or TLR3 agonist (800 ng/mL poly(I:C), pink). Supernatants were collected after 24 hrs and analyzed for type I IFNα (C), IFNβ (D), type II IFNγ (E), type III IFNλ1 (F), IL-6 (G), IL-8 (H), CXCL10 (I) and TNFα (J) expression by ELISA. To evaluate the cytokine response to viral titers and exposure duration, pDCs were exposed to increasing viral inoculums (MOI of 0.01, 0.1 and 1) and IFNα2a mRNA expression was quantified at 24 hrs (K) and IFNα protein secretion at 24, 48, 72 and 96 hrs (L). Graph depicting simple linear regression of IFNα protein with time of exposure (M). Bars and lines represent mean values and symbols represent individual pDC donors (n=3-4). Equal symbols represent equal donors (A-B and C-L). Statistical significance was determined using the ratio paired student T test and compared the treated condition with the time point-matched mock condition (A-L) and simple linear regression (M). *

Article Snippet: To test whether type I IFN contributes to the pDC-mediated inhibition of SARS-CoV-2 inhibition, antibodies blocking the type I IFN receptor (mouse anti-human IFNAR2 antibody, clone MMHAR-2, PBL Assay Science Cat#21385-1) or isotype control (Ultra-LEAF Purified mouse IgG2a, clone MOPC-173, BioLegend Cat#400264) were added to Calu-3 cells in 50 μL PBS and antibodies neutralizing IFNα (mouse anti-human IFN alpha antibody, clone MMHA-2, PBL Assay Science Cat#21100-2) or isotype control (Purified mouse IgG1, clone MOPC-21, BioLegend Cat#400102) were added to 200 μL HSPC-pDC conditioned medium, 10 minutes prior to addition of conditioned medium to the Calu-3 cells.

Techniques: Variant Assay, Expressing, Enzyme-linked Immunosorbent Assay

Journal: bioRxiv

Article Title: Distinct SARS-CoV-2 sensing pathways in pDCs driving TLR7-antiviral vs. TLR2-immunopathological responses in COVID-19

doi: 10.1101/2021.11.23.469755

Figure Lengend Snippet: pDCs were isolated from PBMCs using negative selection and either mock treated (mock) or exposed to SARS-CoV-2 (1 MOI, SARS-2). Supernatants were collected at indicated time points and the production of type I IFNα (A), type III IFNλ1 (B), IL-6 (C), IL-8 (D), CXCL10 (E) and TNFα (F) was quantified by ELISA. Bars and lines represent mean values and symbols represent individual donors (n=3). Statistical significance was determined using the ratio paired student T test by comparing virus exposed cell culture supernatant to the mock control of the matched time point. *

Article Snippet: To test whether type I IFN contributes to the pDC-mediated inhibition of SARS-CoV-2 inhibition, antibodies blocking the type I IFN receptor (mouse anti-human IFNAR2 antibody, clone MMHAR-2, PBL Assay Science Cat#21385-1) or isotype control (Ultra-LEAF Purified mouse IgG2a, clone MOPC-173, BioLegend Cat#400264) were added to Calu-3 cells in 50 μL PBS and antibodies neutralizing IFNα (mouse anti-human IFN alpha antibody, clone MMHA-2, PBL Assay Science Cat#21100-2) or isotype control (Purified mouse IgG1, clone MOPC-21, BioLegend Cat#400102) were added to 200 μL HSPC-pDC conditioned medium, 10 minutes prior to addition of conditioned medium to the Calu-3 cells.

Techniques: Isolation, Selection, Enzyme-linked Immunosorbent Assay, Virus, Cell Culture, Control

Journal: bioRxiv

Article Title: Distinct SARS-CoV-2 sensing pathways in pDCs driving TLR7-antiviral vs. TLR2-immunopathological responses in COVID-19

doi: 10.1101/2021.11.23.469755

Figure Lengend Snippet: To assess whether pDCs could mount protection against SARS-CoV-2, conditioned medium from SARS-CoV-2-exposed pDC cultures (d3 post inoculation with 1 MOI) was added to A549 hACE2 lung epithelial cells (A) or Calu-3 (B) cultures followed by SARS-CoV-2 inoculation. The cell cultures were conditioned with normal medium ( -, grey), pDC supernatant (pDC mock, blue) or SARS-CoV-2-inoculated pDC supernatant (pDC SARS-2, purple), prior to infection with SARS-CoV-2 (0.1 MOI). Supernatants were collected and viral outgrowth was determined 48h post infection. To investigate a potential dose-response, SARS-CoV-2-inoculated pDC supernatant was 3-fold serially diluted prior to addition to Calu-3 cells (purple-pink gradient, B). To determine the involvement of type I IFNs, Calu-3 cells and SARS-CoV-2-inoculated pDC supernatants were pre-treated with antibodies blocking the type I IFN receptor and antibodies neutralizing type I IFNα (IFN-I block) or isotype control antibodies (isot ctrl), prior to the addition of conditioned medium to the cells and infection (C). All antibodies were used at a final concentration of 10 μg/mL. Bars and lines represent median values and symbols represent individual HSPC-pDC donors (n=3-5) Equal symbols represent equal donors. Statistical significance was determined using the ratio paired student T test. *

Article Snippet: To test whether type I IFN contributes to the pDC-mediated inhibition of SARS-CoV-2 inhibition, antibodies blocking the type I IFN receptor (mouse anti-human IFNAR2 antibody, clone MMHAR-2, PBL Assay Science Cat#21385-1) or isotype control (Ultra-LEAF Purified mouse IgG2a, clone MOPC-173, BioLegend Cat#400264) were added to Calu-3 cells in 50 μL PBS and antibodies neutralizing IFNα (mouse anti-human IFN alpha antibody, clone MMHA-2, PBL Assay Science Cat#21100-2) or isotype control (Purified mouse IgG1, clone MOPC-21, BioLegend Cat#400102) were added to 200 μL HSPC-pDC conditioned medium, 10 minutes prior to addition of conditioned medium to the Calu-3 cells.

Techniques: Infection, Blocking Assay, Control, Concentration Assay

Journal: bioRxiv

Article Title: Distinct SARS-CoV-2 sensing pathways in pDCs driving TLR7-antiviral vs. TLR2-immunopathological responses in COVID-19

doi: 10.1101/2021.11.23.469755

Figure Lengend Snippet: Using CRISPR/Cas9, MyD88 knock-out (KO) and AAVS1 KO (control) pDCs were generated. MyD88 protein levels in KO and control pDCs were analyzed by western blotting (A) and cellular DNA was sequenced to perform an Inference of CRISPR Edits (ICE) analysis (B). MyD88 KO and control pDCs were either mock treated (mock) or exposed to SARS-CoV-2 (SARS-2, 1 MOI), supernatants were collected at indicated time points and analyzed for CXCL10 (C) and type I IFNα (D). Type I IFNα production was then determined in cell culture supernatant from SARS-CoV-2 exposed TRIF KO or TRIG+MyD88 KO (E) and RIG-I KO or RIG-I+MyD88 KO (F) pDCs. Bars represent mean values and equal symbols represent equal donors (n=2).

Article Snippet: To test whether type I IFN contributes to the pDC-mediated inhibition of SARS-CoV-2 inhibition, antibodies blocking the type I IFN receptor (mouse anti-human IFNAR2 antibody, clone MMHAR-2, PBL Assay Science Cat#21385-1) or isotype control (Ultra-LEAF Purified mouse IgG2a, clone MOPC-173, BioLegend Cat#400264) were added to Calu-3 cells in 50 μL PBS and antibodies neutralizing IFNα (mouse anti-human IFN alpha antibody, clone MMHA-2, PBL Assay Science Cat#21100-2) or isotype control (Purified mouse IgG1, clone MOPC-21, BioLegend Cat#400102) were added to 200 μL HSPC-pDC conditioned medium, 10 minutes prior to addition of conditioned medium to the Calu-3 cells.

Techniques: CRISPR, Knock-Out, Control, Generated, Western Blot, Cell Culture

Journal: bioRxiv

Article Title: Distinct SARS-CoV-2 sensing pathways in pDCs driving TLR7-antiviral vs. TLR2-immunopathological responses in COVID-19

doi: 10.1101/2021.11.23.469755

Figure Lengend Snippet: Using CRISPR/Cas9, TLR7 knock-out (KO) and AAVS1 KO (control) pDCs were generated and cellular DNA was sequenced for ICE analysis (A). For functional evaluation each KO pDC donor was stimulated with TLR3 (800 ng/mL poly(I:C, pink) or TLR7 (2.5 μg/mL R837, blue) agonist; supernatant was collected after 24 hrs and analyzed for IFNα (B) and CXCL10 (C) protein expression by ELISA. AAVS1 KO and TLR7 KO pDCs were either mock treated (mock) or exposed to SARS-CoV-2 (SARS-2, 1 MOI), supernatants were collected at indicated time points and analyzed for type I IFNα (D) and CXCL10 (E) proteins. Wild type pDCs were exposed to SARS-CoV-2 (0.5 MOI) in the absence or presence of an IRAK4 inhibitor (10 μM), 24 hrs after virus exposure the cell culture supernatants were analyzed for production of type I IFNα, CXCL10 and IL-6 proteins (F). IL-6 protein quantification in AAVS1 KO and TLR7 KO pDCs after SARS-CoV-2 exposure (1 MOI) at indicated time points (G). Bars represent mean values and equal symbols represent the donors used throughout the experiments (n=4). Statistical significance was determined using the ratio paired student T test for agonist or virus treated cells and compared to their respective mock treated conditions, or by unpaired T test when comparing matched conditions between different KOs. *

Article Snippet: To test whether type I IFN contributes to the pDC-mediated inhibition of SARS-CoV-2 inhibition, antibodies blocking the type I IFN receptor (mouse anti-human IFNAR2 antibody, clone MMHAR-2, PBL Assay Science Cat#21385-1) or isotype control (Ultra-LEAF Purified mouse IgG2a, clone MOPC-173, BioLegend Cat#400264) were added to Calu-3 cells in 50 μL PBS and antibodies neutralizing IFNα (mouse anti-human IFN alpha antibody, clone MMHA-2, PBL Assay Science Cat#21100-2) or isotype control (Purified mouse IgG1, clone MOPC-21, BioLegend Cat#400102) were added to 200 μL HSPC-pDC conditioned medium, 10 minutes prior to addition of conditioned medium to the Calu-3 cells.

Techniques: CRISPR, Knock-Out, Control, Generated, Functional Assay, Expressing, Enzyme-linked Immunosorbent Assay, Virus, Cell Culture

Journal: bioRxiv

Article Title: Distinct SARS-CoV-2 sensing pathways in pDCs driving TLR7-antiviral vs. TLR2-immunopathological responses in COVID-19

doi: 10.1101/2021.11.23.469755

Figure Lengend Snippet: Using CRISPR/Cas9, TLR2 KO and AAVS1 KO (control) pDCs were generated, cellular DNA was sequenced for ICE analysis (A) and cells were evaluated functionally by exposure to two different TLR2 agonists; Pam2CSK4 (5 ng/mL, yellow) and Pam3CSK4 (50 ng/mL, orange) (B). Subsequently, AAVS1 KO and TLR2 KO pDCs were either mock treated (mock, grey), exposed to SARS-CoV-2 (SARS-2, 0.5 MOI, purple) or TLR7/8 agonist (2.5 μg/mL R848, red) and supernatants were collected after 24 hrs to quantify type I IFNα (C) and IL-6 (D) protein concentrations. To investigate if pDCs sensed the spike or envelope SARS-CoV-2 proteins, AAVS1 KO and TLR2 KO pDCs were exposed to SARS-CoV-2 (SARS-2, 0.5 MOI, purple) recombinant SARS-CoV-2 spike (S, 1 μg/mL, dark green) or envelope (E, 1 μg/mL, light green) proteins and IL-6 (E) and type I IFNα (F) protein concentrations were quantified. Peripheral blood pDCs were isolated from PBMCs by negative selection and exposed to SARS-CoV-2 (1 MOI, purple), TLR7 agonist (2.5 μg/mL R837, blue) agonist, or E protein (1 μg/mL, light green) for 24 hrs and the concentration of IL-6 (G) and type I IFNα (H) was quantified in cell culture supernatants by ELISA. Bars represent mean values and equal symbols represent equal donors (n=4). Statistical significance was determined using the ratio paired student T test for agonist or virus treated cells and compared to the mock treated condition, or by unpaired T test when comparing matched conditions between different KOs. *

Article Snippet: To test whether type I IFN contributes to the pDC-mediated inhibition of SARS-CoV-2 inhibition, antibodies blocking the type I IFN receptor (mouse anti-human IFNAR2 antibody, clone MMHAR-2, PBL Assay Science Cat#21385-1) or isotype control (Ultra-LEAF Purified mouse IgG2a, clone MOPC-173, BioLegend Cat#400264) were added to Calu-3 cells in 50 μL PBS and antibodies neutralizing IFNα (mouse anti-human IFN alpha antibody, clone MMHA-2, PBL Assay Science Cat#21100-2) or isotype control (Purified mouse IgG1, clone MOPC-21, BioLegend Cat#400102) were added to 200 μL HSPC-pDC conditioned medium, 10 minutes prior to addition of conditioned medium to the Calu-3 cells.

Techniques: CRISPR, Control, Generated, Recombinant, Isolation, Selection, Concentration Assay, Cell Culture, Enzyme-linked Immunosorbent Assay, Virus

Journal: bioRxiv

Article Title: Distinct SARS-CoV-2 sensing pathways in pDCs driving TLR7-antiviral vs. TLR2-immunopathological responses in COVID-19

doi: 10.1101/2021.11.23.469755

Figure Lengend Snippet: The effect of CD304 signaling on IFNα production was tested by incubating pDCs and peripheral blood isolated pDCs (blood pDC) with anti-CD304 antibody (aCD304) or isotype control antibody (isot ctrl) (10 μg/mL) for 15 minutes prior to stimulation with TLR7 agonist (2.5 μg/mL R837). Cell culture supernatants were harvested after 24 hrs to quantify type I IFNα by ELISA (A). Inference of CRISPR Edits (ICE) analysis of CD304 KO pDCs (B). AAVS1 KO and CD304 KO pDCs were either mock treated (mock) or exposed to SARS-CoV-2 (1 MOI), cell culture supernatants were collected at indicated times and analyzed for type I IFNα (C) IL-6 (D) and CXCL10 (E) protein concentrations. Bars and red lines represent mean values, equal symbols represent equal donors (n=4-5). Statistical significance was determined using the ratio paired student T test for agonist or virus treated cells and compared to the time point-matched mock treated condition, or by unpaired T test when comparing matched conditions between different KOs. *

Article Snippet: To test whether type I IFN contributes to the pDC-mediated inhibition of SARS-CoV-2 inhibition, antibodies blocking the type I IFN receptor (mouse anti-human IFNAR2 antibody, clone MMHAR-2, PBL Assay Science Cat#21385-1) or isotype control (Ultra-LEAF Purified mouse IgG2a, clone MOPC-173, BioLegend Cat#400264) were added to Calu-3 cells in 50 μL PBS and antibodies neutralizing IFNα (mouse anti-human IFN alpha antibody, clone MMHA-2, PBL Assay Science Cat#21100-2) or isotype control (Purified mouse IgG1, clone MOPC-21, BioLegend Cat#400102) were added to 200 μL HSPC-pDC conditioned medium, 10 minutes prior to addition of conditioned medium to the Calu-3 cells.

Techniques: Isolation, Control, Cell Culture, Enzyme-linked Immunosorbent Assay, CRISPR, Virus